Structural Biology of Membrane Protein Complexes

Membrane proteins are responsible for many fundamental cellular processes including the transport of ions and metabolites, energy conversion and signal transduction. They are also implicated in a vast range of human diseases. About one third of the human genome encodes for membrane proteins and they are the target of about two thirds of modern pharmaceuticals. However, membrane proteins represent only about 2% of known protein structures in the Protein Data Bank (PDB). This is because membrane proteins are notoriously difficult to express, purify and crystallise for X-ray crystallographic structure determination. In order to progress with our understanding of membrane protein function and to aid the design of relevant drugs, there is an urgent need for the structural characterisation of many more membrane protein families.

We have a long-standing interest in the structural biology of membrane proteins, especially those from the domain of bioenergetics. Most energy in humans is produced in the form of ATP by the mitochondrial respiratory chain consisting of several protein assemblies embedded into lipid membrane (complexes I-V). The main emphasis in the group’s research so far has been on complex I, a huge (up to 1 MDa) enzyme central to cellular energy production, one of the basic foundations of life. Mutations in complex I subunits lead to many human neurodegenerative diseases, and the enzyme is also involved in many common pathologies, including cancer. We have determined all known full atomic structures of complex I, starting from subcomplexes (4-7) and then the entire bacterial enzyme (3), followed recently by the mammalian complex I, the largest asymmetric membrane protein solved to date, with 78 transmembrane helices (1). The structures allowed for the interpretation of over 50 years of functional data. The vast complexity of the enzyme (with up to 9 Fe-S clusters and four proton channels) that belies its efficiency is one of the most intriguing wonders of nature, as the redox energy is used across distances of up to ~300 Å. The structure of the entire complex suggested a uniquely elaborate mechanism of proton translocation, involving long-range conformational changes. We are verifying the mechanism further via structural studies using both X-ray crystallography and single particle cryo-electron microscopy, exploiting the latest advances in the EM hardware and software. In order to build a complete mechanistic understanding of this molecular machine, structural work is supplemented by site-directed mutagenesis and various functional assays using a range of biophysical techniques. We are also expanding our studies to other related complexes, such as Mrp antiporters and membrane-bound hydrogenases, as well as other membrane proteins of interest.

Combined, our studies will move the understanding of redox- and conformationally coupled proton pumps forward and help us derive general and specific features of molecular design in these intricate biological machines, often resembling, but far surpassing in efficiency, human engineering creations. Medical implications are multifaceted, especially for complex I-related diseases, which constitute the most common human genetic disorders.

Contact

Leonid Sazanov

Institute of Science and Technology Austria (IST Austria)
Am Campus 1
A – 3400 Klosterneuburg
Phone: +43 (0)2243 9000-3026
E-mail: sazanov@remove-this.ist.ac.at

CV and publication list

Enquiries from potential post-docs and PhD students are welcome.

Assistant
Rita Six

Phone: +43 (0)2243 9000-1165
E-mail: rita.six@remove-this.ist.ac.at

Team

  • Alexej Charnagalov, Technical Assistant
  • Javier Gutiérrez-Fernández, Postdoc
  • Karol Fiedorczuk, Pre-doctoral Visiting Scientists
  • Kristina Lukic, Scientific Intern
  • Karol Kaszuba, Postdoc
  • James Letts, Postdoc
  • Julia Steiner, PhD Student
  • Margherita Tambalo, Pre-doctoral Visiting Scientists
  • Long Zhou, Postdoc

Selected Projects

- The mechanism of coupling between electron transfer and proton translocation in complex I. Mammalian complex I has a mass of about 1000 kDa and is composed of 44 different subunits (45 in total). The prokaryotic enzyme is simpler (550 kDa, 14 conserved “core” subunits), so we use it as a ‘minimal’ model. It is an L-shaped molecule, formed by hydrophobic and hydrophilic arms. The transfer of two electrons between NADH and quinone, via seven Fe-S clusters in the hydrophilic arm, is coupled to the translocation of four protons across the membrane. How exactly these two spatially separated processes are coupled is not yet clear, although the structure of complex I from T. thermophilus that we have determined suggests that conformational changes propagating along the membrane domain may be involved.

Structure of the entire complex I from T. thermophilus. Each subunit is of different colour and the approximate position of the membrane is indicated. Figure from (3).

We study the coupling mechanism by comparing the X-ray structures of T. thermophilus complex I in different redox states and with various bound substrates. Since crystal contacts may limit any conformational changes, we also study such structures using the latest cryo-EM methods (direct electron detectors and statistical movie processing). We are also collaborating on nano-crystallography using Free Electron Laser sources (such as LCLS at Stanford), which may allow time-resolved studies. Any observed conformational changes will be interpreted mechanistically with the help of site-directed mutagenesis and functional studies.

- Structure of mammalian complex I. Although we have determined the first structure of the bacterial enzyme, the mammalian version contains about 30 additional “accessory” subunits totalling a further ~500 kDa. The function of these proteins, forming a shell around the core subunits, is not clear, although many of them harbour harmful mutations. Using the latest cryo-EM methods, we have now determined the nearly complete atomic structure of the entire mammalian (ovine) complex I at 3.9 Å resolution (1).

Structure of mammalian (ovine) mitochondrial complex I. Two side-views are shown, with core subunits, conserved in bacteria, in grey and “accessory” subunits highlighted in colour. Figure from (1).

Clearly this structure will have far-reaching medical implications, as it provides insight into complex I mechanism, assembly, maturation and dysfunction, allowing detailed molecular analysis of disease-causing mutations. Also, we observe several different conformations of the enzyme, which may be related to active/deactive transitions and so will be studied further in order to define a unified coupling mechanism of complex I.

- Complex I and disease. Since complex I is central to energy production and metabolism, it is intricately involved in many human disorders. We will study the molecular basis of common neurodegenerative diseases, such as LHON, and try to identify potential routes to treatments. Complex I is a major source of reactive oxygen species (ROS) in mitochondria, which can lead to mtDNA damage and possibly plays a role in the aging process. We are developing drug candidates to mitigate such damage. There is increasing evidence for the role of complex I in cancer via ROS signalling. We are collaborating with Pharma and academic partners to help elucidate this role.

- Respiratory supercomplexes. In the mitochondrial membrane in vivo respiratory complexes exist not in isolation but organised into huge “supercomplexes” or “respirasomes” (up to ~ 2 Megadalton in size). Why that is the case is not clear. We have determined several architectures of these supercomplexes, resolved by cryo-EM (2). We describe the arrangement and environment of all the active sites in the component enzymes (complexes I, III and IV). It appears that apart from the stabilisation of individual protein assemblies, one of the functions of supercomplexes may be to prevent excessive production of oxygen radicals. Such radicals are harmful to the DNA and proteins in the cell, and could represent one of the causes of aging.

The architecture of “respirasome”. Complex I is in blue, complex III dimer in green and complex IV in magenta. Redox cofactors are highlighted. Based on the structure determined by cryo-EM at 5.8 Å resolution (2).

- Other membrane protein complexes. We are interested in structure and function of membrane-bound Ni-Fe hydrogenases and Mrp Na+/H+ antiporters, evolutionary ancestors of complex I. They will provide important mechanistic models to compare with complex I. Hydrogenases are also of significant interest to the biofuel industry. Mrp antiporters are essential for the survival of many pathogens, thereby constituting potential antibiotic drug targets.

Selected Publications

    1. Fiedorczuk, K., Letts, J. A., Degliesposti, G., Kaszuba, K., Skehel, M. and Sazanov, L. A. Atomic structure of the entire mammalian mitochondrial complex I. Nature, 538, 406-410, (2016).
    2. Letts, J. A., Fiedorczuk, K. and Sazanov, L. A. The architecture of respiratory supercomplexes. Nature, 537, 644-648, (2016).
    3. Baradaran, R., Berrisford, J.M., Minhas, G. S. and Sazanov, L.A. (2013) Crystal structure of the entire respiratory complex I. Nature, 494, 443-8.
    4. Efremov, R. G. and Sazanov, L. A.  (2011) Structure of the membrane domain of respiratory complex I. Nature, 476, 414-20.
    5. Efremov, R.G., Baradaran, R. and Sazanov, L.A. (2010) The architecture of respiratory complex I, Nature, 465, 441-445.
    6. Sazanov, L.A. and Hinchliffe, P. (2006) Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus.  Science 311, 1430-1436.
    7. Hinchliffe, P. and Sazanov, L.A. (2005) Organization of iron-sulfur clusters in respiratory complex I. Science 309, 771-774.

    Career

    As of 2015 Professor, IST Austria
    2006 Programme leader, MRC Mitochondrial Biology Unit, Cambridge, UK
    2000 Group leader, MRC Mitochondrial Biology Unit, Cambridge, UK
    1997 Research Associate, MRC Laboratory of Molecular Biology, Cambridge, UK
    1994 Research Fellow, Dept. of Biochemistry, Imperial College, London, UK
    1992 Post-doc, School of Biochemistry, University of Birmingham, UK
    1990 Post-doc, Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia
    1990 Ph. D. in Biophysics, Moscow State University, Russia

    Selected Distinctions

    2013 Member of Faculty of 1000
    2012 EMBO Grant
    2009 AMGEN Grant
    2004 Royal Society Grant
    2002 Royal Society Grant
    1992 Wellcome Trust fellowship

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